Adipose-derived stem cells and lattices

ABSTRACT

The present invention provides adipose-derived stem cells and lattices. In one aspect, the present invention provides a lipo-derived stem cell substantially free of adipocytes and red blood cells and clonal populations of connective tissue stem cells. The cells can be employed, alone or within biologically-compatible compositions, to generate differentiated tissues and structures, both in vivo and in vitro. Additionally, the cells can be expanded and cultured to produce hormones and to provide conditioned culture media for supporting the growth and expansion of other cell populations. In another aspect, the present invention provides a lipo-derived lattice substantially devoid of cells, which includes extracellular matrix material from adipose tissue. The lattice can be used as a substrate to facilitate the growth and differentiation of cells, whether in vivo or in vitro, into anlagen or even mature tissues or structures.

This application is a:: 371 of Application One:: PCT/US00/06232 FilingDate:: Mar. 10, 2000 Which is a::NON PROV. OF PROVISIONAL ApplicationTwo:: 60/123,711 Filing Date:: Mar. 10, 1999 And which is a:: NON PROV.OF PROVISIONAL Application Three:: 60/162,462 Filing Date:: Oct. 29,1999

BACKGROUND OF THE INVENTION

In recent years, the identification of mesenchymal stem cells, chieflyobtained from bone marrow, has led to advances in tissue regrowth anddifferentiation. Such cells are pluripotent cells found in bone marrowand periosteum, and they are capable of differentiating into variousmesenchymal or connective tissues. For example, such bone-marrow derivedstem cells can be induced to develop into myocytes upon exposure toagents such as 5-azacytidine (Wakitani et al., Muscle Nerve, 18(12),1417-26 (1995)). It has been suggested that such cells are useful forrepair of tissues such as cartilage, fat, and bone (see, e.g., U.S. Pat.Nos. 5,908,784, 5,906,934, 5,827,740, 5,827,735), and that they alsohave applications through genetic modification (see, e.g., U.S. Pat. No.5,591,625). While the identification of such cells has led to advancesin tissue regrowth and differentiation, the use of such cells ishampered by several technical hurdles. One drawback to the use of suchcells is that they are very rare (representing as few as 1/2,000,000cells), making any process for obtaining and isolating them difficultand costly. Of course, bone marrow harvest is universally painful to thedonor. Moreover, such cells are difficult to culture without inducingdifferentiation, unless specifically screened sera lots are used, addingfurther cost and labor to the use of such stem cells. Thus, there is aneed for a more readily available source for pluripotent stem cells,particularly cells that can be cultured without the requirement forcostly prescreening of culture materials.

Other advances in tissue engineering have shown that cells can be grownin specially-defined cultures to produce three-dimensional structures.Spacial definition typically is achieved by using various acellularlattices or matrices to support and guide cell growth anddifferentiation. While this technique is still in its infancy,experiments in animal models have demonstrated that it is possible toemploy various acellular lattice materials to regenerate whole tissues(see, e.g., Probst et al. BJU Int., 85(3), 362-7 (2000)). A suitablelattice material is secreted extracellular matrix material isolated fromtumor cell lines (e.g, Engelbreth-Holm-Swarm tumor secretedmatrix—“matrigell”). This material contains type IV collagen and growthfactors, and provides an excellent substrate for cell growth (see, e.g.,Vukicevic et al., Exp. Cell Res, 202(1), 1-8 (1992)). However, as thismaterial also facilitates the malignant transformation of some cells(see, e.g., Fridman, et al., Int. J. Cancer, 51(5), 740-44 (1992)), itis not suitable for clinical application. While other artificiallattices have been developed, these can prove toxic either to cells orto patients when used in vivo. Accordingly, there remains a need for alattice material suitable for use as a substrate in culturing andgrowing populations of cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides adipose-derived stem cells and lattices.In one aspect, the present invention provides a lipo-derived stem cellsubstantially free of adipocytes and red blood cells and clonalpopulations of connective tissue stem cells. The cells can be employed,alone or within biologically-compatible compositions, to generatedifferentiated tissues and structures, both in vivo and in vitro.Additionally, the cells can be expanded and cultered to produce hormonesand to provide conditioned culture media for supporting the growth andexpansion of other cell populations. In another aspect, the presentinvention provides a lipo-derived lattice substantially devoid of cells,which includes extracellular matrix material from adipose tissue. Thelattice can be used as a substrate to facilitate the growth anddifferentiation of cells, whether in vivo or in vitro, into anlagen oreven mature tissues or structures.

Considering how plentiful adipose tissue is, the inventive cells andlattice represent a ready source of pluripotent stem cells. Moreover,because the cells can be passaged in culture in an undifferentiatedstate under culture conditions not requiring prescreened lots of serum,the inventive cells can be maintained with considerably less expensethan other types of stem cells. These and other advantages of thepresent invention, as well as additional inventive features, will beapparent from the accompanying drawings and in the following detaileddescriptions.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention pertains to a lipo-derived stem cell.Preferably, the stem cell is substantially free of other cell types(e.g., adipocytes, red blood cells, other stromal cells, etc.) andextracellular matrix material; more preferably, the stem cell iscompletely free of such other cell types and matrix material.Preferably, the inventive cell is derived from the adipose tisue of aprimate, and more preferably a higher primate (e.g, a baboon or ape).Typically, the inventive cell will be derived from human adipose tissue,using methods such as described herein.

While the inventive cell can be any type of stem cell, for use in tissueengineering, desirably the cell is of mesodermal origin. Typically suchcells, when isolated, retain two or more mesodermal or mesenchymaldevelopmental phenotypes (i.e., they are pluripotent). In particular,such cells generally have the capacity to develop into mesodermaltissues, such as mature adipose tissue, bone, various tissues of theheart (e.g., pericardium, epicardium, epimyocardium, myocardium,pericardium, valve tissue, etc.), dermal connective tissue, hemangialtissues (e.g., corpuscles, endocardium, vascular epithelium, etc.),muscle tissues (including skeletal muscles, cardiac muscles, smoothmuscles, etc.), urogenital tissues (e.g., kidney, pronephros, meta- andmeso-nephric ducts, metanephric diverticulum, ureters, renal pelvis,collecting tubules, epithelium of the female reproductive structures(particularly the oviducts, uterus, and vagina)), pleural and peritonealtissues, viscera, mesodermal glandular tissues (e.g., adrenal cortextissues), and stromal tissues (e.g., bone marrow). Of course, inasmuchas the cell can retain potential to develop into mature cells, it alsocan realize its developmental phenotypic potential by differentiatinginto an appropriate precursor cell (e.g., a preadipocyte, a premyocyte,a preosteocyte, etc.). Also, depending on the culture conditions, thecells can also exhibit developmental phenotypes such as embryonic,fetal, hematopoetic, neurogenic, or neuralgiagenic developmentalphenotypes. In this sense, the inventive cell can have two or moredevelopmental phenotypes such as adipogenic, chondrogenic, cardiogenic,dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic,neurogenic, neuralgiagenic, urogenitogenic, osteogenic, pericardiogenic,peritoneogenic, pleurogenic, splanchogenic, and stromal developmentalphenotypes. While such cells can retain two or more of thesedevelopmental phenotypes, preferably, such cells have three or more suchdevelopmental phenotypes (e.g, four or more mesodermal or mesenchymaldevelopmental phenotypes), and some types of inventive stem cells have apotential to acquire any mesodermal phenotppe through the process ofdifferentiation.

The inventive stem cell can be obtained from adipose tissue by anysuitable method. A first step in any such method requires the isolationof adipose tissue from the source animal. The animal can be alive ordead, so long as adipose stromal cells within the animal are viable.Typically, human adipose stromal cells are obtained from living donors,using well-recognized protocols such as surgical or suction lipectomy.Indeed, as liposuction procedures are so common, liposuction effluent isa particularly preferred source from which the inventive cells can bederived.

However derived, the adipose tissue is processed to separate stem cellsfrom the remainder of the material. In one protocol, the adipose tissueis washed with physiologically-compatible saline solution (e.g.,phosphate buffered saline (PBS)) and then vigorously agitated and leftto settle, a step that removes loose mater (e.g., damaged tissue, blood,erythrocytes, etc.) from the adipose tissue. Thus, the washing andsettling steps generally are repeated until the supernatant isrelatively clear of debris.

The remaining cells generally will be present in lumps of various size,and the protocol proceeds using steps gauged to degrade the grossstructure while minimizing damage to the cells themselves. One method ofachieving this end is to treat the washed lumps of cells with an enzymethat weakens or destroys bonds between cells (e.g., collagenase,dispase, trypsin, etc.). The amount and duration of such enzymatictreatment will vary, depending on the conditions employed, but the useof such enzymes is generally known in the art. Alternatively or inconjunction with such enzymatic treatment, the lumps of cells can bedegraded using other treatments, such as mechanical agitation, sonicenergy, thermal energy, etc. If degradation is accomplished by enzymaticmethods, it is desirable to neutralize the enzyme following a suitableperiod, to minimize deleterious effects on the cells.

The degradation step typically produces a slurry or suspension ofaggregated cells (generally liposomes) and a fluid fraction containinggenerally free stromal cells (e.g., red blood cells, smooth musclecells, endothelial cells, fibroblast cells, and stem cells). The nextstage in the separation process is to separate the aggregated cells fromthe stromal cells. This can be accomplished by centrifugation, whichforces the stromal cells into a pellet covered by supernatant. Thesupernatant then can be discarded and the pellet suspended in aphysiologically-compatible fluid. Moreover, the suspended cellstypically include erythrocytes, and in most protocols it is desirable tolyse these. Methods for selectively lysing erythrocytes are known in theart, and any suitable protocol can be employed (e.g., incubation in ahyper- or hypotonic medium). Of course, if the erythrocytes are lysed,the remaining cells should then be separated from the lysate, forexample by filtration or centrifugation. Of course, regardless ofwhether the erythrocytes are lysed, the suspended cells can be washed,re-centrifuged, and resuspended one or more successive times to achievegreater purity. Alteratively, the cells can be separated using a cellsorter or on the basis of cell size and granularity, stem cells beingrelatively small and agranular. Expression of telomerase can also serveas a stem cell-specific marker. They can also be separatedimmunohistochemically, for example, by panning or using magnetic beads.Any of the steps and procedures for isolating the inventive cells can beperformed manually, if desired. Alteratively, the process of isolatingsuch cells can be facilitated through a suitable device, many of whichare known in the art (see, e.g., U.S. Pat. No. 5,786,207).

Following the final isolation and resuspension, the cells can becultured and, if desired, assayed for number and viability to assess theyield. Desirably the cells can be cultured without differentiation usingstandard cell culture media (e.g, DMEM, typically supplemented with5-15% (e.g., 10%) serum (e.g., fetal bovine serum, horse serum, etc.).Preferably, the cells can be passaged at least five times in such mediumwithout differentiating, while still retaining their developmentalphenotype, and more preferably, the cells can be passaged at least 10times (e.g., at least 15 times or even at least 20 times) without losingdevelopmental phenotype. Thus, culturing the cells of the presentinvention without inducing differentiation can be accomplished withoutspecially screened lots of serum, as is generally the case formesenchymal stem cells (e.g., derived from marrow). Methods formeasuring viability and yield are known in the art (e.g., trypan blueexclusion).

Following isolation, the stem cells are further separated by phenotypicidentification, to identify those cells that have two or more of theaforementioned developmental phenotypes. Typically, the stromal cellsare plated at a desired density such as between about 100 cells/cm² toabout 100,000 cells/cm² (such as about 500 cells/cm² to about 50,000cells/cm^(2,) or, more particuarly, between about 1,000 cells/cm² toabout 20,000 cells/cm²). If plated at lower densities (e.g., about 300cells/cm²), the cells can be more easily clonally isolated. For example,after a few days, cells plated at such densities will proliferate into apopulation.

Such cells and populations can be clonally expanded, if desired, using asuitable method for cloning cell populations. For example, aproliferated population of cells can be physically picked and seededinto a separate plate (or the well of a multi-well plate).Alternatively, the cells can be subcloned onto a multi-well plate at astatistical ratio for facilitating placing a single cell into each well(e.g., from about 0.1 to about 1 cell/well or even about 0.25 to about0.5 cells/well, such as 0.5 cells/well). Of course, the cells can becloned by plating them at low density (e.g, in a petri-dish or othersuitable substrate) and isolating them from other cells using devicessuch as a cloning rings. Alternatively, where an irradiation source isavailable, clones can be obtained by permitting the cells to grow into amonolayer and then shielding one and irradiating the rest of cellswithin the monolayer. The surviving cell then will grow into a clonalpopulation. While production of a clonal population can be expanded inany suitable culture medium, a preferred culture condition for cloningstem cells (such as the inventive stem cells or other stem cells) isabout ⅔ F₁₂medium+20% serum (preferably fetal bovine serum) and about ⅓standard medium that haw been conditioned with stromal cells (e.g.,cells from the stromal vascular fraction of liposuction aspirate), therelative proportions being determined volumetrically).

In any event, whether clonal or not, the isolated cells can be culturedto a suitable point when their developmental phenotype can be assessed.As mentioned, the inventive cells have at least two of theaforementioned developmental phenotypes. Thus, one or more cells drawnfrom a given clone can be treated to ascertain whether it possesses suchdevelopmental potentials. One type of treatment is to culture theinventive cells in culture media that has been conditioned by exposureto mature cells (pr precursors thereof) of the respective type to bedifferentiated (e.g., media conditioned by exposure to myocytes caninduce myogenic differentiation, media conditioned by exposure to heartvalve cells can induce differentiation into heart valve tissue, etc.).Of course, defined media for inducing differentiation also can beemployed. For example, adipogenic developmental phenotype can beassessed by exposing the cell to a medium that facilitates adipogenesis,e.g., containing a glucocorticoid (e.g., isobutyl-methylxanthine,dexamethasone, hydrocortisone, cortisone, etc.), insulin, a compoundwhich elevates intracellular levels of cAMP (e.g., dibutyryl-cAMP,8-CPT-cAMP (8-(4)chlorophenylthio)-adenosine 3′, 5′cyclic monophosphate;8-bromo-cAMP; dioctanoyl-cAMP, forskolin etc.), and/or a compound whichinhibits degradation of cAMP (e.g., a phosphodiesterase inhibitor suchas methyl isobutylxanthine, theophylline, caffeine, indomethacin, andthe like). Thus, exposure of the stem cells to between about 1 μM andabout 10 μM insulin in combination with about 10⁻⁹ M to about 10⁻⁶ M to(e.g., about 1 μM) dexamethasone can induce adipogenic differentiation.Such a medium also can include other agents, such as indomethicin (e.g.,about 100 μM to about 200 μM, if desired, and preferably the medium isserum free. Osteogenic developmental phenotype can be assessed byexposing the cells to between about 10⁻⁷ M and about 10⁻⁹ Mdexamethasone (e.g., about 1 μM) in combination with about 10 μM toabout 50 μM ascorbate-2-phosphate and between about 10 nM and about 50nM β-glycerophosphate, and the medium also can include serum (e.g.,bovine serum, horse serum, etc.). Myogenic differentiation can beinduced by exposing the cells to between about 10 μM and about 100 μMhydrocortisone, preferably in a serum-rich medium (e.g., containingbetween about 10% and about 20% serum (either bovine, horse, or amixture thereof)). Chondrogenic differentiation can be induced byexposing the cells to between about 1 μM to about 10 μM insulin andbetween about 1 μM to about 10 μM transferrin, between about 1 ng/ml and10 ng/ml transforming growth factor (TGF) β1, and between about 10 nMand about 50 nM ascorbate-2-phosphate (50 nM). For chondrogenicdifferentiation, preferably the cells are cultured in high density(e.g., at about several million cells/ml or using micromass culturetechniques), and also in the presence of low amounts of serum (e.g.,from about 1% to about 5%). The cells also can be induced to assume adevelopmentally more immature phenotype (e.g., a fetal or embryonicphenotype). Such induction is achieved upon exposure of the inventivecell to conditions that mimic those within fetuses and embryos. Forexample, the inventive cells or populations can be co-cultured withcells isolated from fetuses or embryos, or in the presence of fetalserum. Along these lines, the cells can be induced to differentiate intoany of the aforementioned mesodermal lineages by co-culturing them withmature cells of the respective type, or precursors thereof. Thus, forexample, myogenic differentiation can be induced by culturing theinventive cells with myocytes or precursors, and similar results can beachieved with respect to the other tissue types mentioned herein. Othermethods of inducing differentiation are known in the art, and many ofthem can be employed, as appropriate.

After culturing the cells in the differentiating-inducing medium for asuitable time (e.g., several days to a week or more), the cells can beassayed to determine whether, in fact, they have differentiated toacquire physical qualities of a given type of cell. One measurement ofdifferentiation per se is telomere length, undifferentiated stem cellshaving longer telomeres than differentiated cells; thus the cells can beassayed for the level of telomerase activity. Alternatively, RNA orproteins can be extracted from the cells and assayed (via Northernhybridization, rtPCR, Western blot analysis, etc.) for the presence ofmarkers indicative of the desired phenotype. Of course, the cells can beassayed immunohistochemically or stained, using tissue-specific stains.Thus, for example, to assess adipogenic differentiation, the cells canbe stained with fat-specific stains (e.g., oil red O, safarin red, sudanblack, etc.) or probed to assess the presence of adipose-related factors(e.g., type IV collagen, PPAR-γ, adipsin, lipoprotein lipase, etc.).Similarly, ostogenesis can be assessed by staining the cells withbone-specific stains (e.g., alkaline phosphatase, von Kossa, etc.) orprobed for the presence of bone-specific markers (e.g., osteocalcin,osteonectin, osteopontin, type I collagen, bone morphogenic proteins,cbfa, etc.). Myogensis can be assessed by identifyng classicalmorphologic changes (e.g., polynucleated cells, syncitia formation,etc.), or assessed biochemically for the presence of muscle-specificfactors (e.g., myo D, myosin heavy chain, NCAM, etc.). Chondrogenesiscan be determined by staining the cells using cartallge-specific stains(e.g., alcian blue) or probing the cells for the expression/productionof cartilage-specific molecules (e.g., sulfated glycosaminoglycans andproteoglycans (e.g., keratin, chondroitin, etc.) in the medium, type IIcollagen, etc.). Other methods of assessing developmental phenotype areknown in the art, and any of them is appropriate. For example, the cellscan be sorted by size and granularity. Also, the cells can be used togenerate monoclonal antibodies, which can then be employed to assesswhether they preferentially bind to a given cell type. Correlation ofantigenicity can confirm that the stem cell has differentiated along agiven developmental pathway.

While the cell can be solitary and isolated from other cells, preferablyit is within a population of cells, and the invention provides a definedpopulation including the inventive cell. In some embodiments, thepopulation is heterogeneous. Thus, for example, the population caninclude support cells for supplying factors to the inventive cells. Ofcourse, the inventive stem cells can themselves serve as support cellsfor culturing other types of cells (such as other types of stem cells,e.g., as neural stem cells (NSC), hematopoetic stem cells (HPC,particularly CD34⁺ stem cells), embryonic stem cells (ESC) and mixturesthereof), and the population can include such cells. In otherembodiments, the population is substantially homogeneous, consistingessentially of the inventive lipo-derived stem cells.

As the inventive cells can be cloned, a substantially homogeneouspopulation containing them can be clonal. Indeed, the invention alsopertains to any defined clonal cell population consisting essentially ofmesodermal stem cells, connective tissue stem cell, or mixtures thereof.In this embodiment, the cells can be lipo-derived or derived from othermesodermal or connective cell tissues (e.g., bone marrow, muscle, etc.)using methods known in the art. After the isolation, the cells can beexpanded clonally as described herein.

The inventive cells (and cell populations) can be employed for a varietyof purposes. As mentioned, the cells can support the growth andexpansion of other cell types, and the invention pertains to methods foraccomplishing this. In one aspect, the invention pertains to a method ofconditioning culture medium using the inventive stem cells and toconditioned medium produced by such a method. The medium becomesconditioned upon exposing a desired culture medium to the cells underconditions sufficient for the cells to condition it. Typically, themedium is used to support the growth of the inventive cells, whichsecrete hormones, cell matrix material, and other factors into themedium. After a suitable period (e.g., one or a few days), the culturemedium containing the secreted factors can be separated from the cellsand stored for future use. Of course, the inventive cells andpopulations can be re-used successively to condition medium, as desired.In other applications (e.g., for co-culturing the inventive cells withother cell types), the cells can remain within the conditioned medium.Thus, the invention provides a conditioned medium obtained using thismethod, which either can contain the inventive cells or be substantiallyfree of the inventive cells, as desired.

The conditioned medium can be used to support the growth and expansionof desired cell types, and the invention provides a method of culturingcells (particularly stem cells) using the conditioned medium. The methodinvolves maintaining a desired cell in the conditioned medium underconditions for the cell to remain viable. The cell can be maintainedunder any suitable condition for culturing them, such as are known inthe art. Desirably, the method permits successive rounds of mitoticdivision of the cell to form an expanded population. The exactconditions (e.g., temperature, CO₂ levels, agitation, presence ofantibiotics, etc.) will depend on the other constituents of the mediumand on the cell type. However, optimizing these parameters are withinthe ordinary skill in the art. In some embodiments, it is desirable forthe medium to be substantially free of the lipo-derived cells employedto condition the medium as described herein. However, in otherembodiments, it is desirable for the lipo-derived cells to remain in theconditioned medium and co-cultured with the cells of interest. Indeed,as the inventive lipo-derived cells can express cell-surface mediatorsof intercellular communication, it often is desirable for the inventivecells and the desired other cells to be co-cultured under conditions inwhich the two cell types are in contact. This can be achieved, forexample, by seeding the cells as a heterogeneous population of cellsonto a suitable culture substrate. Alternatively, the inventivelipo-derived cells can first be grown to confluence, which will serve asa substrate for the second desired cells to be cultured within theconditioned medium.

In another embodiment, the inventive lipo-derived cells can begenetically modified, e.g., to express exogenous genes or to repress theexpression of endogenous genes, and the invention provides a method ofgenetically modifying such cells and populations. In accordance withthis method, the cell is exposed to a gene transfer vector comprising anucleic acid including a transgene, such that the nucleic acid isintroduced into the cell under conditions appropriate for the transgeneto be expressed within the cell. The transgene generally is anexpression cassette, including a coding polynucleotide operably linkedto a suitable promoter. The coding polynucleotide can encode a protein,or it can encode biologically active RNA (e.g., antisense RNA or aribozyme). Thus, for example, the coding polynucleotide can encode agene conferring resistance to a toxin, a hormone (such as peptide growthhormones, hormone releasing factors, sex hormones, adrenocorticotrophichormones, cytokines (e.g., interferins, interleukins, lymphokines),etc.), a cell-surface-bound intracellular signaling moiety (e.g., celladhesion molecules, hormone receptors, etc.), a factor promoting a givenlineage of differentiation, etc. Of course, where it is desired toemploy gene transfer technology to deliver a given transgene, itssequence will be known.

Within the expression cassette, the coding polynucleotide is operablylinked to a suitable promoter. Examples of suitable promoters includeprokaryotic promoters and viral promoters (e.g., retrovial ITRs, LTRs,immediate early viral promoters (IEp), such as herpesvirus IEp (e.g.,ICP4-IEp and ICP0-IEp), cytomegalovirus (CMV) IEp, and other viralpromoters, such as Rous Sarcoma Virus (RSV) promoters, and MurineLeukemia Virus (MLV) promoters). Other suitable promoters are eukaryoticpromoters, such as enhancers (e.g., the rabbit β-globin regulatoryelements), constitutively active promoters (e.g., the β-actin promoter,etc.), signal specific promoters (e.g., inducible promoters such as apromoter responsive to RU486, etc.), and tissue-specific promoters. Itis well within the skill of the art to select a promoter suitable fordriving gene expression in a predefined cellular context. The expressioncassette can include more than one coding polynucleotide, and it caninclude other elements (e.g., polyadenylation sequences, sequencesencoding a membrane-insertion signal or a secretion leader, ribosomeentry sequences, transcriptional regulatory elements (e.g., enhancers,silencers, etc.), and the like), as desired.

The expression cassette containing the transgene should be incorporatedinto a genetic vector suitable for delivering the transgene to thecells. Depending on the desired end application, any such vector can beso employed to genetically modify the cells (e.g., plasmids, naked DNA,viruses such as adenovirus, adeno-associated visus, herpesviruses,lentiviruses, papillomaviruses, retroviruses, etc.). Any method ofconstructing the desired expression cassette within such vectors can beemployed, many of which are well known in the art (e.g., direct cloning,homologous recombination, etc.). Of course, the choice of vector willlargely determine the method used to introduce the vector into the cells(e.g., by protoplast fusion, calcium-phosphate precipitation, gene gun,electroporation, infection with viral vectors, etc.), which aregenerally known in the art.

The genetically altered cells can be employed as bioreactors forproducing the product of the transgene. In other embodiments, thegenetically modified cells are employed to deliver the transgene and itsproduct to an animal. For example, the cells, once genetically modified,can be introduced into the animal under conditions sufficient for thetransgene to be expressed in vivo.

In addition to serving as useful targets for genetic modification, manycells and populations of the present invention secrete hormones (e.g.,cytokines, peptide or other (e.g., monobutyrin) growth factors, etc.).Some of the cells naturally secrete such hormones upon initialisolation, and other cells can be genetically modified to secretehormones, as discussed herein. The cells of the present invention thatsecrete hormones can be used in a variety of contexts in vivo and invitro. For example, such cells can be employed as bioreactors to providea ready source of a given hormone, and the invention pertains to amethod of obtaining hormones from such cells. In accordance with themethod, the cells are cultured, under suitable conditions for them tosecrete the hormone into the culture medium. After a suitable period oftime, and preferably periodically, the medium is harvested and processedto isolate the hormone from the medium. Any standard method (e.g., gelor affinity chromatography, dialysis, lyophilization, etc.) can be usedto purify the hormone from the medium, many of which are known in theart.

In other embodiments, cells (and populations) of the present inventionsecreting hormones can be employed as therapeutic agents. Generally,such methods involve transferring the cells to desired tissue, either invitro (e.g., as a graft prior to implantation or engrafting) or in vivo,to animal tissue directly. The cells can be transferred to the desiredtissue by any method appropriate, which generally will vary according tothe tissue type. For example, cells can be transferred to a graft bybathing the graft (or infusing it) with culture medium containing thecells. Alternatively, the cells can be seeded onto the desired sitewithin the tissue to establish a population. Cells can be transferred tosites in vivo using devices such as catherters, trocars, cannulae,stents (which can be seeded with the cells), etc. For theseapplications, preferably the cell secretes a cytokine or growth hormonesuch as human growth factor, fibroblast growth factor, nerve growthfactor, insulin-like growth factors, hemopoetic stem cell growthfactors, members of the fibroblast growth factor family, members of theplatelet-derived growth factor family, vascular and endothelial cellgrowth factors, members of the TGFb family (including bone morphogenicfactor), or enzymes specific for congenital disorders (e.g.,distrophin).

In one application, the invention provides a method of promoting theclosure of a wound within a patient using such cells. In accordance withthe method, the inventive cells secreting the hormone are transferred tothe vicinity of a wound under conditions sufficient for the cell toproduce the hormone. The presence of the hormone in the vicinity of thewound promotes closure of the wound. The method promotes closure of bothexternal (e.g., surface) and internal wounds. Wounds to which thepresent inventive method is useful in promoting closure include, but arenot limited to, abrasions, avulsions, blowing wounds, burn wounds,contusions, gunshot wounds, incised wounds, open wounds, penetratingwounds, perforating wounds, puncture wounds, seton wounds, stab wounds,surgical wounds, subcutaneous wounds, or tangential wounds. The methodneed not achieve complete healing or closure of the wound; it issufficient for the method to promote any degree of wound closure. Inthis respect, the method can be employed alone or as an adjunct to othermethods for healing wounded tissue.

Where the inventive cells secrete an angiogenic hormone (e.g., vasculargrowth factor, vascular and endothelial cell growth factor, etc.), they(as well as populations containing them) can be employed to induceangiogenesis within tissues. Thus, the invention provides a method ofpromoting neovascularization within tissue using such cells. Inaccordance with this method, the cell is introduced the desired tissueunder conditions sufficient for the cell to produce the angiogenichormone. The presence of the hormone within the tissue promotesneovascularization within the tissue.

Because the inventive stem cells have a developmental phenotype, theycan be employed in tissue engineering. In this regard, the inventionprovides a method of producing animal matter comprising maintaining theinventive cells under conditions sufficient for them to expand anddifferentiate to form the desired matter. The matter can include maturetissues, or even whole organs, including tissue types into which theinventive cells can differentiate (as set forth herein). Typically, suchmatter will comprise adipose, cartilage, heart, dermal connectivetissue, blood tissue, muscle, kidney, bone, pleural, splanchnic tissues,vascular tissues, and the like. More typically, the matter will comprisecombinations of these tissue types (i.e., more than one tissue type).For example, the matter can comprise all or a portion of an animal organ(e.g., a heart, a kidney) or a limb (e.g., a leg, a wing, an arm, ahand, a foot, etc.). Of course, in as much as the cells can divide anddifferentiate to produce such structures, they can also form anlagen ofsuch structures. At early stages, such anlagen can be cryopreserved forfuture generation of the desired mature structure or organ.

To produce such structures, the inventive cells and populations aremaintained under conditions suitable for them to expand and divide toform the desired structures. In some applications, this is accomplishedby transferring them to an animal (i.e., in vivo) typically at a sightat which the new matter is desired. Thus, for example, the invention canfacilitate the regeneration of tissues (e.g., bone, muscle, cartilage,tendons, adipose, etc.) within an animal where the cells are implantedinto such tissues. In other embodiments, and particularly to createanlagen, the cells can be induced to differentiate and expand intotissues in vitro. In such applications, the cells are cultured onsubstrates that facilitate formation into three-dimensional structuresconducive for tissue development. Thus, for example, the cells can becultured or seeded onto a bio-compatible lattice, such as one thatincludes extracellular matrix material, synthetic polymers, cytokines,growth factors, etc. Such a lattice can be molded into desired shapesfor facilitating the development of tissue types. Also, at least at anearly stage during such culturing, the medium and/or substrate issupplemented with factors (e.g., growth factors, cytokines,extracellular matrix material, etc.) that facilitate the development ofappropriate tissue types and structures. Indeed, in some embodiments, itis desired to co-culture the cells with mature cells of the respectivetissue type, or precursors thereof, or to expose the cells to therespective conditioned medium, as discussed herein.

To facilitate the use of the inventive lipo-derived cells andpopulations for producing such animal matter and tissues, the inventionprovides a composition including the inventive cells (and populations)and biologically compatible lattice. Typically, the lattice is formedfrom polymeric material, having fibers as a mesh or sponge, typicallywith spaces on the order of between about 100 μm and about 300 μm. Sucha structure provides sufficient area on which the cells can grow andproliferate. Desirably, the lattice is biodegradable over time, so thatit will be absorbed into the animal matter as it develops. Suitablepolymeric lattices, thus, can be formed from monomers such as glycolicacid, lactic acid, propyl fumarate, caprolactone, hyaluronan, hyaluronicacid, and the like. Other lattices can include proteins,polysaccharides, polyhydroxy acids, polyorthoesthers, polyanhydrides,polyphosphazenes, or synthetic polymers (particularly biodegradablepolymers). Of course, a suitable polymer for forming such lattice caninclude more than one monomers (e.g., combinations of the indicatedmonomers). Also, the lattice can also include hormones, such as growthfactors, cytokines, and morphogens (e.g., retinoic acid, aracadonicacid, etc.), desired extracellular matrix molecules (e.g., fibronectin,laminin, collagen, etc.), or other materials (e.g., DNA, viruses, othercell types, etc.) as desired.

To form the composition, the cells are introduced into the lattice suchthat they permeate into the interstitial spaces therein. For example,the matrix can be soaked in a solution or suspension containing thecells, or they can be infused or injected into the matrix. Aparticularly preferred composition is a hydrogel formed by crosslinkingof a suspension including the polymer and also having the inventivecells dispersed therein. This method of formation permits the cells tobe dispersed throughout the lattice, facilitating more even permeationof the lattice with the cells. Of course, the composition also caninclude mature cells of a desired phenotppe or precursors thereof,particularly to potentate the induction of the inventive stem cells todifferentiate appropriately within the lattice (e.g., as an effect ofco-culturing such cells within the lattice).

The composition can be employed in any suitable manner to facilitate thegrowth and generation of the desired tissue types, structures, oranlagen. For example, the composition can be constructed usingthree-dimensional or sterotactic modeling techniques. Thus, for example,a layer or domain within the composition can be populated by cellsprimed for osteogenic differentiation, and another layer or domainwithin the composition can be populated with cells primed for myogenicand/or chondrogenic development. Bringing such domains intojuxtaposition with each other facilitates the molding anddifferentiation of complex structures including multiple tissue types(e.g., bone surrounded by muscle, such as found in a limb). To directthe growth and differentiation of the desired stucture, the compositioncan be cultured ex vivo in a bioreactor or incubator, as appropriate. Inother embodiments, the structure is implanted within the host animaldirectly at the site in which it is desired to grow the tissue orstructure. In still another embodiment, the composition can be engraftedon a host (typically an animal such as a pig, baboon, etc.), where itwill grow and mature until ready for use. Thereafter, the maturestructure (or anlage) is excised from the host and implanted into thehost, as appropriate.

Lattices suitable for inclusion into the composition can be derived fromany suitable source (e.g., matrigel), and some commercial sources forsuitable lattices exist (e.g., suitable of polyglycolic acid can beobtained from sources such as Ethicon, N.J.). Another suitable latticecan be derived from the acellular portion of adipose tissue—i.e.,adipose tissue extracellular matrix matter substantially devoid ofcells, and the invention provides such a lipo-derived lattice.Typically, such lipo-derived lattice includes proteins such asproteoglycans, glycoproteins, hyaluronins, fibronectins, collagens (typeI, type II, type III, type IV, type V, type VI, etc.), and the like,which serve as excellent substrates for cell growth. Additionally, suchlipo-derived lattices can include hormones, preferably cytokines andgrowth factors, for facilitating the growth of cells seeded into thematrix.

The lipo-derived matrix can be isolated form adipose tissue similarly asdescribed above, except that it will be present in the acellularfraction. For example, adipose tissue or derivatives thereof (e.g., afraction of the cells following the centrifugation as discussed above)can be subjected to sonic or thermal energy and/or enzymatic processedto recover the matrix material. Also, desirably the cellular fraction ofthe adipose tissue is disrupted, for example by treating it withlipases, detergents, proteases, and/or by mechanical or sonic disruption(e.g., using a homogenizer or sonicator). However isolated, the materialis initially identified as a viscous material, but it can besubsequently treated, as desired, depending on the desired end use. Forexample, the raw matrix material can be treated (e.g., dialyzed ortreated with proteases or acids, etc.) to produce a desirable latticematerial. Thus the lattice can be prepared in a hyrated form or it canbe dried or lyophilized into a substantially anhydrous form or a powder.Therafter, the powder can be rehydrated for use as a cell culturesubstrate, for example by suspending it in a suitable cell culturemedium. In this regard, the lipo-derived lattice can be mixed with othersuitable lattice materials, such as described above. Of course, theinvention pertains to compositions including the lipo-derived latticeand cells or populations of cells, such as the inventive lipo-derivedcells and other cells as well (particularly other types of stem cells).

As discussed above, the cells, populations, lattices, and compositionsof the invention can be used in tissue engineering and regeneration.Thus, the invention pertains to an implantable structure (i.e., animplant) incorporating any of these inventive features. The exact natureof the implant will vary according to the use to which it is to be put.The implant can be or comprise, as described, mature tissue, or it caninclude immature tissue or the lattice. Thus, for example, one type ofimplant can be a bone implant, comprising a population of the inventivecells that are undergoing (or are primed for) osteogenicdifferentiation, optionally seeded within a lattice of a suitable sizeand dimension, as described above. Such an implant can be injected orengrafted within a host to encourage the generation or regeneration ofmature bone tissue within the patient. Similar implants can be used toencourage the growth or regeneration of muscle, fat, cartilage, tendons,etc., within patients. Other types of implants are anlagen (such asdescribed herein), e.g., limb buds, digit buds, developing kidneys, etc,that, once engrafted onto a patient, will mature into the appropriatestructures.

The lipo-derived lattice can conveniently be employed as part of a cellculture kit. Accordingly, the invention provides a kit including theinventive lipo-derived lattice and one or more other components, such ashydrating agents (e.g., water, physiologically-compatible salinesolutions, prepared cell culture media, serum or derivatives thereofetc.), cell culture substrates (e.g., culture dishes, plates, vials,etc.), cell culture media (whether in liquid or powdered form),antibiotic compounds, hormones, and the like. While the kit can includeany such ingredients, preferably it includes all ingredients necessaryto support the culture and growth of desired cell types upon propercombination. Of course, if desired, the kit also can include cells(typically frozen), which can be seeded into the lattice as describedherein.

While many aspects of the invention pertain to tissue growth anddifferentiation, the invention has other applications as well. Forexample, the lipo-derived lattice can be used as an experimentalreagent, such as in developing improved lattices and substrates fortissue growth and differentiation. The lipo-derived lattice also can beemployed cosmetically, for example, to hide wrinkles, scars, cutaneousdepressions, etc., or for tissue augmentation. For such applications,preferably the lattice is stylized and packaged in unit dosage form. Ifdesired, it can be admixed with carriers (e.g., solvents such asglycerine or alcohols), perfumes, antibiotics, colorants, and otheringredients commonly employed in cosmetic products. The substrate alsocan be employed autologously or as an allograft, and it can used as, orincluded within, ointments or dressings for facilitating wound healing.The lipo-dived cells can also be used as experimental reagents. Forexample, they can be employed to help discover agents responsible forearly events in differentiation. For example, the inventive cells can beexposed to a medium for inducing a particular line of differentiationand then assayed for differential expression of genes (e.g., byrandom-primed PCR or electrophoresis or protein or RNA, etc.).

As any of the steps for isolating the inventive stem cells or thelipo-derived lattice, the, the invention provides a kit for isolatingsuch reagents from adipose tissues. The kit can include a means forisolating adipose tissue from a patient (e.g., a cannula, a needle, anaspirator, etc.), as well as a means for separating stromal cells (e.g.,through methods described herein). The kit can be employed, for example,as a bedside source of stem cells that can then be re-introduced fromthe same individual as appropriate. Thus, the kit can facilitate theisolation of lipo-derived stem cells for implantation in a patientneeding regrowth of a desired tissue type, even in the same procedure.In this respect, the kit can also include a medium for differentiatingthe cells, such as those set forth herein. As appropriate, the cells canbe exposed to the medium to prime them for differentiation within thepatient as needed. Of course, the kit can me used as a convenient sourceof stem cells for in vitro manipulation (e.g., cloning ordifferentiating as described herein). In another embodiment, the kit canbe employed for isolating a lipo-derived lattice as described herein.

EXAMPLES

While one of skill in the art is fully able to practice the instantinvention upon reading the foregoing detailed description, the followingexamples will help elucidate some of its features. In particular, theydemonstrate the isolation of a human lipo-derived stem cellsubstantially free of mature adipocytes, the isolation of a clonalpopulation of such cells, the ability of such cells to differentiate invivo and in vitro, and the capacity of such cells to support the growthof other types of stem cells. The examples also demonstrate theisolation of a lipo-derived lattice substantially free of cells that iscapable of serving as a suitable substrate for cell culture. Of course,as these examples are presented for purely illustrative purposes, theyshould not be used to construe the scope of the invention in a limitedmanner, but rather should be seen as expanding upon the foregoingdescription of the invention as a whole.

The procedures employed in these examples, such as surgery, cellculture, enzymatic digestion, histology, and molecular analysis ofproteins and polynucleotides, are familiar to those of ordinary skill inthis art. As such, and in the interest of brevity, experimental detailsare not recited in detail.

Example 1

This example demonstrates the isolation of a human lipo-derived stemcell substantially free of mature adipocytes.

Raw liposuction aspirate was obtained from patients undergoing electivesurgery. Prior to the liposuction procedures, the patients were givenepinephrine to minimize contamination of the aspirate with blood. Theaspirate was strained to separate associated adipose tissue pieces fromassociated liquid waste. Isolated tissue was rinsed thoroughly withneutral phosphate buffered saline and then enzymatically dissociatedwith 0.075% w/v colagenase at 37° C. for about 20 minutes underintermittent agitation. Following the digestion, the collagenase wasneutralized, and the slurry was centrifuged at about 260 g for about 10minutes, which produced a multi-layered supernatant and a cellularpellet. The supernatant was removed and retained for further use, andthe pellet was resuspended in an erythrocyte-lysing solution andincubated without agitation at about 25° C. for about 10 minutes.Following incubation, the medium was neutralized, and the cells wereagain centrifuged at about 250 g for about 10 minutes. Following thesecond centrifugation, the cells were suspended, and assessed forviability (using trypan blue exclusion) and cell number. Thereafter,they were plated at a density of about at about 1×10⁶ cells/100 mm dish.They were cultured at 37° C. in DMEM+fetal bovine serum (about 10%) inabout 5% CO₂.

The majority of the cells were adherent, small, mononucleic, relativelyagranular fibroblast-like cells containing no visible lipid droplets.The majority the cells stained negatively with oil-red O and von Kossa.The cells were also assayed for expression of telomerase (using acommercially available TRAP assay kit), using HeLa cells and HN-12 cellsas positive controls. Human foreskin fibroblasts and HN-12 heated cellextracts were used as negative controls. Telomeric products wereresolved onto a 12.5% polyacrylamide cells and the signals determined byphosphorimaging. Telemeric ladders representing telomerase activity wereobserved in the adipose-derived stem cells as well as the positivecontrols. No ladders were observed in the negative controls.

Thus, these cells were not identifiable as myocytes, adipocytes,chondrocytes, osteocytes, or blood cells. These results demonstrate thatthe adipose-derived cells express telomerase activity similar to thatpreviously reported for human stem cells.

Subpopulations of these cells were then exposed to the following mediato assess their developmental phenotype:

Adipogenesis Osteogenesis Myogenesis Chondrogenesis DMEM DMEM DMEM DMEM10% FBS 10% FBS 10% FBS 1% FBS 0.5 mM IBMX 5% horse serum 5% horse serum6.25 μg/ml insulin 1 μM dexamethasone 0.1 μM dexamethasone 50 μMhydrocortisone 6.25 μg/ml transferrin 10 μM insulin 50 μM ascorbate-2-1% ABAM 10 ng/ml TGFβ1 200 μM indomethacin phosphate 50 nM ascorbate-2-1% ABAM 10 mM β- phosphate glycerophosphate 1% ABAM 1% ABAM

A population was cultured at high density in the chondrogenic medium forseveral weeks. Histological analysis of the issue culture and paraffinsections was performed with H&E, alcian blue, toludene blue, andGoldner's trichrome staining at 2, 7, and 14 days. Immunohistochemistrywas performed using antibodies against chondroitin-4-sulfate and keratinsulfate and type II collagen. Qualitative estimate of matrix stainingwas also performed. The results indicated that cartilaginous spheroidnodules with a distinct border of perichondral cells formed as early as48 hours after initial treatment. Untreated control cells exhibited noevidence of chondrogenic differentiation. These results confirm that thestem cells have chondrogenic developmental phenotype.

A population was cultured until near confluence and then exposed to theadipogenic medium for several weeks. The population was examined at twoand four weeks after plating by colorimetric assessment of relativeopacity following oil red-O staining. Adipogenesis was determined to beunderway at two weeks and quite advanced at four weeks (relative opacityof 1 and 5.3, respectively). Bone marrow-derived stem cells wereemployed as a positive control, and these cells exhibited slightly lessadipogenic potential (relative density of 0.7 and 2.8, respectively).

A population was cultured until near confluence and then exposed to theosteogeneic medium for several weeks. The population was examined at twoand four weeks after plating by colorimetric assessment of relativeopacity following von Kossa staining. Osteogenesis was determined to beunderway at two weeks and quite advanced at four weeks (relative opacityof 1.1 and 7.3, respectively. Bone marrow-derived stem cells wereemployed as a positive control, and these cells exhibited slightly lessosteogenic potential (relative density of 0.2 and 6.6, respectively).

A population was cultured until near confluence and then exposed to themyogenic medium for several weeks. The population was examined at one,three, and six weeks after plating by assessment of multinucleated cellsand expressin of muscle-specific proteins (MyoD and myosin heavy chain).Human foreskin fibroblasts and skeletal myoblasts were used as controls.Cells expressing MyoD and myosin were found at all time points followingexposure to the myogenic medium in the stem cell population, and theproportion of such cells increased at 3 and 6 weeks. Multinucleatedcells were observed at 6 weeks. In contrast, the fibroblasts exhibitednone of these characteristics at any time points.

These results demonstrate the isolation of a human lipo-derivedpluripotent stem cell substantially free of mature adipocytes.

Example 2

This example demonstrates that lipo-derived stem cells do notdifferentiate in response to 5-azacytidine.

Lipo-derived stem cells obtained in accordance with Example 1 werecultured in the presence of 5-azacytidine. In contrast with bonemarrow-derived stem cells, exposure to this agent did not inducemyogenic differentiation (see Wakitani et al., supra).

Example 3

This example demonstrates the generation of a clonal population of humanlipo-derived stem cells.

Cells isolated in accordance with the procedure set forth in Example 1were plated at about 5,000 cells/100 mm dish and cultured for a few daysas indicated in Example 1. After some rounds of cell division, someclones were picked with a cloning ring and transferred to wells in a 48well plate. These cells were cultured for several weeks, changing themedium twice weekly, until they were about 80% to about 90% confluent(at 37° C. in about 5% CO₂ in ⅔ F₁₂medium+20% fetal bovine serum and ⅓standard medium that was first conditioned by the cells isolated inExample 1, “cloning medium”). Thereafter, each culture was transferredto a 35 mm dish and grown, and then retransferred to a 100 mm dish andgrown until close to confluent. Following this, one cell population wasfrozen, and the remaining populations were plated on 12 well plates, at1000 cells/well.

The cells were cultured for more than 15 passages in cloning medium andmonitored for differentiation as indicated in Example 1. Theundifferentiated state of each clone remained true after successiverounds of differentiation.

Populations of the clones then were established and exposed toadipogenic, chondrogenic, myogenic, and osteogenic medium as discussedin Example 1. It was observed that at least one of the clones was ableto differentiate into bone, fat, cartilage, and muscle when exposed tothe respective media, and most of the clones were able to differentiateinto at least three types of tissues. The capacity of the cells todevelop into muscle and cartilage further demonstrates thepluripotentiality of these lipo-derived stem cells.

These results demonstrate that the lipo-derived stem cells can bemaintained in an undifferentiated state for many passages without therequirement for specially pre-screened lots of serum. The results alsodemonstrate that the cells retain pluripotentiality following suchextensive passaging, proving that the cells are indeed stem cells andnot merely committed progenitor cells.

Example 4

This example demonstrates the lipo-derived stem cells can support theculture of other types of stem cells.

Human lipo-derived stem cells were passaged onto 96 well plates at adensity of about 30,000/well, cultured for one week and then irradiated.Human CD34⁺ hematopoetic stem cells isolated from umbilical cord bloodwere then seeded into the wells. Co-cultures were maintained inMyeloCult H5100 media, and cell viability and proliferation weremonitored subjectively by microscopic observation. After two weeks ofco-culture, the hematopoetic stem cells were evaluated for CD34expression by flow cytometry.

Over a two-week period of co-culture with stromal cells, thehematopoetic stem cells formed large colonies of rounded cells. Flowanalysis revealed that 62% of the cells remained CD34+. Based onmicroscopic observations, human adipo-derived stromal cells maintainedthe survival and supported the growth of human hematopoetic stem cellsderived from umbilical cord blood.

These results demonstrate that stromal cells from human subcutaneousadipose tissue are able to support the ex vivo maintenance, growth anddifferentiation of other stem cells.

Example 5

This example demonstrates that the lipo-derived stem cells candifferentiate in vivo.

Four groups (A-D) of 12 athymic mice each were implanted subcutaneouslywith hydroxyapatite/tricalcium phosphate cubes containing the following:Group A contained lipo-derived stem cells that had been pretreated withosteogenic medium as set forth in Example 1. Group B contained untreatedlipo-derived stem cells. Group C contained osteogenic medium but nocells. Group D contained non-osteogenic medium and no cells. Within eachgroup, six mice were sacrificed at three weeks, and the remaining micesacrificed at eight weeks following implantation. The cubes wereextracted, fixed, decalcified, and sectioned. Each section was analyzedby staining with H&E, Mallory bone stain, and immunostaining forosteocalcin.

Distinct regions of osteoid-like tissue staining for osteocalcin andMallory bone staining was observed in sections from groups A and B.Substantially more osteoid tissue was observed in groups A and B than inthe other groups (p<0.05 ANOVA), but no significant difference inosteogenesis was observed between groups A and B. Moreover, aqualitative increase in bone growth was noted in both groups A and Bbetween 3 and 8 weeks. These results demonstrate that the lipo-derivedstem cells can differentiate in vitro.

Example 6

This example demonsrates the isolation of a lipo-derived latticesubstantially devoid of cells.

In one protocol withheld supernatant from Example 1 was subjected toenzymatic digestion for three days in 0.05% trypsin EDTA/100 U/mldeoxyribonuclease to destroy the cells. Every day the debris was rinsedin saline and fresh enzyme was added. Thereafter the material was rinsedin saline and resuspended in 0.05% collagease and about 0.1% lipase topartially digest the proteins and fat present. This incubation continuedfor two days.

In another protocol, the withheld supernatant from Example 1 wasincubated in EDTA to eliminate any epithelial cells. The remaining cellswere lysed using a buffer containing 1% NP40, 0.5% sodium deoxycholate,0.1% SDS, 5 mM EDTA, 0.4M NaCl, 50 mMTris-HCL (pH 8) and proteaseinhibitors, and 10 μg/ml each of leupeptin, chymostatin, antipain, andpepstatin A. Finally, the tissue was extensively washed in PBS withoutdivalent cations.

After both preparatory protocols, remaining substance was washed andidentified as a gelatinous mass. Microscopic analysis of this materialrevealed that it contained no cells, and it was composed of high amountsof collagen (likely type IV) and a wide variety of growth factors.Preparations of this material have supported the growth of cells,demonstrating it to be an excellent substrate for tissue culture.

Incorporation by Reference

All sources (e.g., inventor's certificates, patent applications,patents, printed publications, repository accessions or records, utilitymodels, world-wide web pages, and the like) referred to or citedanywhere in this document or in any drawing, Sequence Listing, orStatement filed concurrently herewith are hereby incorporated into andmade part of this specification by such reference thereto.

Guide to Interpretation

The foregoing is an integrated description of the invention as a whole,not merely of any particular element of facet thereof. The descriptiondescribes “preferred embodiments” of this invention, including the bestmode known to the inventors for carrying it out. Of course, upon readingthe foregoing description, variations of those preferred embodimentswill become obvious to those of ordinary skill in the art The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law.

As used in the foregoing description and in the following claims,singular indicators (eg., “a” or “one”) include the plural, unlessotherwise indicated. Recitation of a range of discontinuous values isintended to serve as a shorthand method of referring individually toeach separate value falling within the range, and each separate value isincorporated into the specification as if it were individually listedAdditionally, the following terms are defined as follows:

An anlage is a primordial structure that has a capacity to develop intoa specific mature structure.

A developmental phenotype is the potential of a cell to acquire aparticular physical phenotype through the process of differentiation.

A hormone is any substance that is secreted by a cell and that causes aphenotypic change in the same or another cell upon contact.

A stem cell is a pluripotent cell that has the capacity to differentiatein accordance with at least two discrete developmental pathways.

As regards the claims in particular, the term “consisting essentiallyof” indicates that unlisted ingredients or steps that do not materiallyaffect the basic and novel properties of the invention can be employedin addition to the specifically recited ingredients or steps. Incontrast, terms such as “comprising,” “having,” and “including” indicatethat any ingredients or steps can be present in addition to thoserecited. The term “consisting of” indicates that only the recitedingredients or steps are present, but does not foreclose the possibilitythat equivalents of the ingredients or steps can substitute for thosespecifically recited.

We claim:
 1. An isolated adipose-derived stem cell that candifferentiate into two or more of the group consisting of a bone cell, acartilage cell, a nerve cell, or a muscle cell.
 2. An isolated,adipose-derived multipotent cell that differentiates into cells of twoor more mesodermal phenotypes.
 3. An isolated adipose-derived stem cellthat differentiates into two or more of the group consisting of a fatcell, a bone cell, a cartilage cell, a nerve cell, or a muscle cell. 4.An isolated adipose-derived stem cell that differentiates into acombination of any of a fat cell, a bone cell, a cartilage cell, a nervecell, or a muscle cell.
 5. A substantially homogeneous population ofadipose-derived stem cells, comprising a pluality of the stem cell ofclaim 1, 3 or
 4. 6. The adipose-derived stem cell of claim 1, 3 or 4which can be cultured for at least 15 passages without differentiating.7. The adipose-derived stem cell of claim 1, 3 or 4 which is human. 8.The cell of any of claim 1, 3 or 4 which is genetically modified.
 9. Thecell of any of claim 1, 3 or 4, which has a cell-surface boundintercellular signaling moiety.
 10. The cell of any of claim 1, 3 or 4,which secretes a hormone.